Slidders’ Fuchsin-Miller for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Slidders’ Fuchsin-Miller

for Fibrin

steps

materials

print

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Fuchsin
Material Amount
Acid fuchsin 1 g
Acetic acid, glacial 2.5 mL
Distilled water 100 mL
Miller
Material Amount
Milling yellow 3G 2.5 g
2-ethoxy-ethanol 100 mL
Phosphotungstic acid
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL

Material	Amount
Acid fuchsin	1	g
Acetic acid, glacial	2.5	mL
Distilled water	100	mL

Material	Amount
Milling yellow 3G	2.5	g
2-ethoxy-ethanol	100	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate or B5 overnight. Paraffin process overnight. Overnight formalin fixation and paraffin processing can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
If mercury fixed, remove with the iodine-thiosulphate sequence.
If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
Rinse well with distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in fuchsin for 10 minutes.
Rinse with distilled water.
Differentiate with phosphotungstic acid for 5 minutes.
Rinse well with distilled water.
Blot.
Rinse well with cellosolve.
Place into milling yellow until fibrin is red and tissues are yellow.
This step takes from ½ – 4 hours.
Rinse briefly with 1% aqueous acetic acid.
Rinse with cellosolve.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – red
Other tissue – yellow
Nuclei – black

Notes

It is very important that the milling yellow solution be completely anhydrous. Even small amounts of water or other solvents can cause problems with displacement and incomplete removal of red dye. For that reason, step 12 should be thorough and the milling yellow should be used in a Coplin jar with a lid sealed with tape.
A well stained section would show red fibrin only, muscle and erythrocytes being yellow. With poorly fixed material, both erythrocytes and muscle fibres may resist displacement of the red dye and they may have some residual red colouring. Sometimes this may be overcome by prestaining either with saturated picric acid in absolute ethanol or the MSB martius yellow solution for two minutes immediately before step 7, but a better resolution is correct fixation and processing.
Some intracellular materials may stain red, such as Paneth cell granules.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Martius, Scarlet and Blue (MSB) for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Martius, Scarlet and Blue (MSB)

for Fibrin

steps

materials

print

The MSB (Martius, Scarlet and Blue) method for fibrin is a reliable technique. It is more automatic than other methods, i.e. it is less dependent on skill and experience and is consequently suitable for a routine laboratory. Overnight mercuric chloride fixation (formol sublimate or B5) is preferred, followed by overnight paraffin processing, although formalin fixed, paraffin embedded material can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections, as for the Picro-Mallory.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Martius
Material Amount
Martius yellow 0.5 g
Phosphotungstic acid 2 g
Ethanol, 95% 100 mL
Scarlet
Material Amount
Crystal scarlet 1 g
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Blue
Material Amount
Methyl blue 0.5 g
Acetic acid, glacial 1 mL
Distilled water 98 mL
Phosphotungstic acid
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL

Material	Amount
Martius yellow	0.5	g
Phosphotungstic acid	2	g
Ethanol, 95%	100	mL

Material	Amount
Crystal scarlet	1	g
Acetic acid, glacial	2.5	mL
Distilled water	97.5	mL

Material	Amount
Methyl blue	0.5	g
Acetic acid, glacial	1	mL
Distilled water	98	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Overnight formalin fixation is usually satisfactory, but avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
If mercury fixed, remove with the iodine-thiosulphate sequence.
If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
Rinse well in distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in martius yellow for 2 minutes.
Rinse with distilled water.
Place in crystal scarlet for 10 minutes.
Differentiate with phosphotungstic acid until only fibrin is red (up to 10 minutes).
Place in methyl blue until collagen is blue (up to 10 minutes).
Rinse briefly with 1% aqueous acetic acid.
Dehydrate rapidly with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – red
Fresh fibrin – yellow
Erythrocytes – yellow
Connective tissue – blue

Notes

Crystal scarlet is more commonly known as ponceau 6R.
Steps 10 & 11 specify the time as up to. These times should be established by inspection, but will generally remain consistent.
Lendrum’s recommendation for formalin fixed material was to dewax sections and treat with trichlorethylene in a sealed contained for 48 hours at 56°C, then to refix in absolute ethanol saturated with both picric acid and mercuric chloride for 24 hours before proceeding to step 2. The alternative treatment with Bouin’s fluid given at step 3 is often satisfactory.
Bancroft notes that dyes other than those originally given may be used. Some may not be easily available, and no CI numbers were given.
Color Dye Options
Yellow Lissamine fast yellow
Blue Durazol blue
Pontamine sky blue
Fast green FCF
Naphthalene black 10B
Red Ponceau de xylidine
Azofuchsin

Color	Dye Options
Yellow	Lissamine fast yellow
Blue	Durazol blue Pontamine sky blue Fast green FCF Naphthalene black 10B
Red	Ponceau de xylidine Azofuchsin

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Obadiah for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Obadiah

for Fibrin

steps

materials

print

The name of this stain comes from the letters OBDR45, which refer to the dyes used: Orange, Blue, Direct Red 45 (a synonym for one of the red dyes).

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Results are usually poor with formalin fixed material, even if treated with Bouin’s fluid at 56°C for an hour or so.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Picro-mercuric ethanol
Material Amount
Ethanol, absolute 100 mL
Picric acid to saturation
Mercuric chloride to saturation
Orange
Material Amount
Orange G 0.5 g
Phosphotungstic acid 1 g
Ethanol, 95% 100 mL
Blue
Material Amount
Naphthalene blue black CS 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Red – Option 1
Material Amount
Chicago red 2.5 g
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Red – Option 2
Material Amount
Polar brilliant red BN 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Ethanol, absolute	100	mL
Picric acid	to	saturation
Mercuric chloride	to	saturation

Material	Amount
Orange G	0.5	g
Phosphotungstic acid	1	g
Ethanol, 95%	100	mL

Material	Amount
Naphthalene blue black CS	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Chicago red	2.5	g
Acetic acid, glacial	2.5	mL
Distilled water	97.5	mL

Material	Amount
Polar brilliant red BN	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, as this produces tissues that stain poorly. Sections should be 3-5 µ thick.

Protocol

Dewax sections with xylene.
Place into a sealed container of trichlorethylene at 56°C for 48 hours.
Rinse well with absolute ethanol.
Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
Remove mercury pigment with the iodine-thiosulphate sequence.
Stain nuclei with an acid resistant nuclear stain.
Place in the orange stain for 2 minutes.
Rinse with distilled water.
Place in the Blue stain up to 30 minutes.
Differentiate with the polyacid for 5 minutes.
Place in the Red stain 15-20 minutes (polar brilliant red) or 15-30 minutes (chicago red).
Rinse with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Old fibrin – black
Younger fibrin – may be yellow
Connective tissue – red

Notes

Note that this method reverses the usual order and uses a blue stain before the red stain.
Lendrum considered that this method demonstrated fibrin that other methods stained as collagen.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Picro-Mallory for Fibrin – Long Version

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Picro-Mallory

for Fibrin – Long Version

steps

materials

print

Lendrum, Fraser and others published several fibrin stains, the most well known being the Picro-Mallory. There are several variants of the method, some more complicated than others.

See this alternative protocol for a shorter version.

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Adequate results can be obtained with thorough formalin fixation (24 hours), thorough processing (overnight), sectioning, followed by refixing in Bouin’s fluid at 56°C for an hour or so. However, results are inferior to those obtained by the full procedure. It is also a method where experience is required for optimal results.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the
celestine blue-hemalum sequence.
Yellow mordant
Material Amount
Picric acid, sat. in 80% ethanol 100 mL
Orange G 0.2 g
Lissamine yellow 0.2 g
Stock differentiator
Material Amount
Picric acid 2.5 g
Ethanol, 95% 100 mL
Phosphotungstic acid 25 g
Dissolve ingredients and filter.
Red differentiator
Material Amount
Stock differentiator 40 mL
Ethanol, 95% 20 mL
Distilled water 90 mL
Blue differentiator
Material Amount
Stock differentiator 10 mL
Distilled water 90 mL
Red stain
Material Option 1 Option 2 Option 3
Acid fuchsin 1 g – 0.4 g
Lissamine fast red – – 0.2 g
Biebrich scarlet – 1 g –
Acetic acid, glacial 1 mL 1 mL 1 mL
Distilled water 99 mL 99 mL 99 mL
Blue stain
Material Amount
Methyl blue 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Picric acid, sat. in 80% ethanol	100	mL
Orange G	0.2	g
Lissamine yellow	0.2	g

Material	Amount
Picric acid	2.5	g
Ethanol, 95%	100	mL
Phosphotungstic acid	25	g

Material	Amount
Stock differentiator	40	mL
Ethanol, 95%	20	mL
Distilled water	90	mL

Material	Amount
Stock differentiator	10	mL
Distilled water	90	mL

Material	Option 1	Option 2	Option 3
Acid fuchsin	1	g	–	0.4	g
Lissamine fast red	–	–	0.2	g
Biebrich scarlet	–	1	g	–
Acetic acid, glacial	1	mL	1	mL	1	mL
Distilled water	99	mL	99	mL	99	mL

Material	Amount
Methyl blue	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, especially with formalin fixatives, as this produces tissues that stain poorly even with secondary fixation such as Bouin’s fluid at 56°C for an hour. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment with the iodine-thiosulphate sequence.
Stain nuclei with an acid resistant nuclear stain.
Place in yellow mordant for 2-3 minutes.
Wash in distilled water until only erythrocytes are yellow.
Place in the red stain for 5-10 minutes.
Rinse with 1% aqueous acetic acid.
Differentiate with the red differentiator until fibrin is prominent microscopically.
Rinse well in distilled water.
Place in the blue stain for 5 minutes.
Rinse briefly with 1% aqueous acetic acid.
Place in the blue differentiator for 1-2 minutes.
Biefly rinse with 1% aqueous acetic acid.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – clear red
Erythrocytes – yellow
Connective tissue – blue

Notes

The most common dye combination is acid fuchsin and methyl blue.
Adequate results may be obtained with 24-48 hours fixation in neutral buffered formalin, overnight paraffin processing and secondary fixation of sections with Bouin’s picro-formol-acetic for 1 hour at 56°C.
Good results are obtained with the fixation and processing recommended in the text.
Optimal results are obtained with the fixation and processing recommended in the text, followed by degreasing and secondary fixation in picro-mercuric ethanol.
Replace step 1 with the following:
1. Dewax sections with xylene.
2. Place in trichlorethylene in a sealed container at 56°C for 48 hours.
3. Rinse sections well with absolute ethanol.
4. Place in picro-mercuric-ethanol for 24 hours.
5. Wash sections with water, and continue from step 2.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Yellowsolve I for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Yellowsolve I

for Fibrin

steps

materials

print

Materials

Celestine blue-hemalum nuclear stain, or similar.
Trichlorethylene
Tetrachlorethylene
Solution A
Material Amount
Phloxine B 0.5 g
Calcium chloride 0.5 g
Distilled water 100 mL
Solution B
Material Amount
Lissamine flavine FF to saturation
2-Ethoxyethanol 50 mL
Ethyl phosphate 50 mL
Optional – an elastic fibre stain: Orcein or Weigert’s iron resorcin fuchsin, or variant.

Material	Amount
Phloxine B	0.5	g
Calcium chloride	0.5	g
Distilled water	100	mL

Material	Amount
Lissamine flavine FF	to saturation
2-Ethoxyethanol	50	mL
Ethyl phosphate	50	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Protocol

Dewax sections with xylene.
Place in trichlorethylene for 24 to 48 hours.
Bring sections to water.
Stain elastic fibres if desired.
Stain nuclei with the celestine blue-hemalum sequence.
Wash well with water.
Place in solution A for 30 minutes.
Rinse with 2-ethoxyethanol then with tetrachlorethylene.
Place into tetrachlorethylene overnight.
Rinse with 2-ethoxyethanol.
Differentiate with solution B, controlling microscopically.
Rinse with 2-ethoxyethanol.
Clear in xylene and mount with a synthetic resinous medium.

Expected Results

Fibrin – red
Background – yellow
Nuclei – black
Acidophil cytoplasmic inclusions – red

Notes

2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH₃CH₂OCH₂CH₂OH.
Tetrachlorethylene has the formula Cl₂C=CCl₂ or C₂Cl₄
Trichlorethylene has the formula ClHC=CCl₂ or C₂HCl₃
Both trichlorethylene and tetrachlorethylene should be used in a fume hood.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Masson’s HES General Oversight Stain

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Masson's HES

General Oversight Stain

steps

materials

print

This is also known as the Hematoxylin Erythrosine Saffron stain.

Materials

Regressive hemalum
Solution A
Material Amount
Erythrosine B 1 g
Tap water 100 mL
Dissolve the erythrosine B and filter. Preserve with a few drops drops of chloroform.
Solution B
Material Amount
Saffron 2 g
Distilled water 100 mL
Strong formalin 1 mL
Tannic acid, 5% aqueous 1 mL
Add the saffron stigmata to the water and heat in a boiling water bath for one hour. Filter, and add the formalin and tannic acid. Life is a few weeks.

Material	Amount
Erythrosine B	1	g
Tap water	100	mL

Material	Amount
Saffron	2	g
Distilled water	100	mL
Strong formalin	1	mL
Tannic acid, 5% aqueous	1	mL

Tissue Sample

No particular fixative was specified. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

Bring sections to water with xylene and ethanol.
Treat with Bouin’s fluid if desired.
Stain nuclei with hemalum, differentiate and blue.
Wash well with water.
Place in solution A for 2-5 minutes.
Wash rapidly with tap water.
Differentiate erythrosine with 70% ethanol for a few seconds to decolorize collagen.
Wash rapidly with tap water.
Place into solution B for 5 minutes.
Wash rapidly with tap water.
Dehydrate rapidly with absolute ethanol.
Clear with xylene and mount with a synthetic resinous medium.

Expected Results

Nuclei – blue
Cytoplasm – red shades
Muscle – pink
Elastic fibres – pink
Collagen – orange-yellow

Notes

Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
Eosin B or phloxine B may be substituted for erythrosine B.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Biological Staining Methods, 6th ed. (1957)
Gurr, G. T.,
George T. Gurr, London, UK
Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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HPS Variant General Oversight Stain

By Carmen WongProtocols, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

HPS Variant

General Oversight Stain

steps

materials

print

This is also known as the Hematoxylin Phloxine Saffron stain.

Materials

Regressive hemalum
Solution A
Material Amount
Phloxine B 2 g
Distilled water 100 mL
Dissolve the phloxine B and filter. Preserve with a crystal of thymol.
Solution B
Material Amount
Saffron 30 g
Absolute ethanol 1 L
Add the saffron to 500ml absolute ethanol and extract at 60°C for 48 hours. Decant, and store in a dark bottle. Repeat with 500 mL fresh absolute ethanol and combine with the first extraction.

Material	Amount
Phloxine B	2	g
Distilled water	100	mL

Material	Amount
Saffron	30	g
Absolute ethanol	1	L

Tissue Sample

No fixative was specified, but most methods using acid dyes benefit from picric acid or mercuric chloride fixation. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

Bring sections to water with xylene and ethanol.
Treat with hemalum for 5 minutes, differentiate and blue.
Wash with running tap water for 5 minutes.
Place in solution A for 5 minutes.
Wash with running tap water for 5 minutes.
Differentiate phloxine with 95% ethanol.
Thoroughly dehydrate with absolute ethanol.
Place into solution B for 5-10 minutes until a suitable red-yellow balance is achieved.
Dehydrate with 4 changes of absolute ethanol.
Clear with 4 changes of xylene and mount with a synthetic resinous medium.

Expected Results

Nuclei – blue
Cytoplasm – red shades
Collagen – yellow

Notes

Harris’ hemalum was named, but the formula given was a variant of Bencosme’s hemalum.
Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
Store and use the saffron solution at room temperature. It has a limited life of about a month and is best when freshly made. It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied. If contaminated with moisture the solution must be discarded.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Histonet posting

March 30, 2023

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Engel & Cunningham’s Trichrome for Muscle Fibres, Types I & II

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, One-Step

Protocol

Engel & Cunningham's Trichrome

for Muscle Fibres, Types I & II

steps

materials

print

Materials

Harris alum hematoxylin
Solution A
Material Amount
Chromotrope 2R 0.6 g
Fast green FCF 0.3 g
Phosphotungstic acid 0.6 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Adjust to pH 3.4 with 1N NaOH.
Solution B
Material Amount
Acetic acid, glacial 0.2 mL
Distilled water 100 mL

Material	Amount
Chromotrope 2R	0.6	g
Fast green FCF	0.3	g
Phosphotungstic acid	0.6	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Acetic acid, glacial	0.2	mL
Distilled water	100	mL

Tissue Sample

Unfixed frozen sections of liquid nitrogen quenched muscle. Sections should be 5-10µ in thickness. The fibres should be in cross section, as close to a right angle to their length as possible.

Protocol

Cut cryostat sections and dry at room temperature.
Stain nuclei with Harris’ hematoxylin for 5 minutes.
Rinse well with distilled water.
Place into solution A for 10 minute.
Rinse with solution B for a few seconds to differentiate.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Type I fibres – green with a red cast
Type II fibres – green

Notes

Both fibre types are green overall, but type I fibres contain more red staining granular material and have a red cast.
This is a modification of Gomori’s trichrome.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques, Ed. 3
Butterworth, London, UK.
Citing:
Engel & Cunningham, (1963)
Rapid examination of muscle tissue.
An improved trichrome method for fresh frozen biopsy specimens.
Neurology, vol.13, pp.921

March 30, 2023

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Papanicolaou’s Alcoholic Trichrome for Exfoliated Cells

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Papanicolaou's Alcoholic Trichrome

for Exfoliated Cells

steps

materials

print

This is the Papanicolaou technique commonly used to screen for cervical cancer.

Materials

Ehrlich’s or Harris’ alum hematoxylin or equivalent
Solution A (OG6)
Material Amount
Orange G 5 g
Phosphotungstic acid 0.15 g
Ethanol, 95% 1 L

Material	Amount
Orange G	5	g
Phosphotungstic acid	0.15	g
Ethanol, 95%	1	L

Solution B

Material	Variant
Material	EA 25		EA 36		EA 50		EA 65
Light green SF	2.2	g	2.25	g	0.45	g	1.125	g
Bismarck brown	0.6	g	0.5	g	0.5	g	0.5	g
Eosin Y	2.2	g	2.25	g	2.25	g	2.25	g
Phosphotungstic acid	1.7	g	2	g	2	g	2	g
Lithium carbonate	5	mg	5	mg	5	mg	5	mg
Ethanol, 95%	1	L	1	L	1	L	1	L

Preparation

For each variant:

Dissolve the bismarck brown in 100 mL ethanol, the light green in 450 mL ethanol, and the eosin Y in another 450 mL ethanol.
Combine the three solutions.
Add the acid and lithium carbonate.
Filter.

Tissue Sample

Ethanol-ether fixed smears of exfoliated epithelial cells.

Protocol

Bring smears to water via graded ethanols.
Stain nuclei with alum hematoxylin for 2 minutes.
Blue.
Rinse with 95% ethanol.
Place into solution A for 1 minute.
Rinse with 95% ethanol.
Place into solution B for 2 minutes.
Rinse well 95% ethanol.
Dehydrate rapidly with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Cells – shades of orange, pink or green

Notes

Although the method calls for smears fixed in an equal parts mixture of ethanol and diethyl ether, smears fixed in 95% or absolute ethanol are completely satisfactory.
Lithium carbonate saturates in water at 1% at room temperature, so 0.5 mL contains 5 mg.
EA 65 is recommended for specimens with large amounts of mucus.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Papanicolaou, (1942)
Science, v.95, pp.438
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Duprès’ Trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Duprès' Trichrome

for Muscle and Collagen

steps

materials

print

Materials

Gallego’s carbol fuchsin or
Duprès’ magenta
Solution A
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution B
Material Amount
Toluidine blue 0.25 g
Orange G 4 g
Oxalic acid 4 g
Distilled water 100 mL

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Material	Amount
Toluidine blue	0.25	g
Orange G	4	g
Oxalic acid	4	g
Distilled water	100	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from mercuric chloride fixation. Treatment with picric acid may be less satisfactory due to the use of the basic dye, toluidine blue.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with either Gallego’s carbol fuchsin or Duprès’ magenta
Wash with water.
Place into solution A for 10 minute.
Rinse with distilled water.
Place into solution B for 1-5 minutes.
Drain.
Place into 95% ethanol until differentiated.
Dehydrate with 100% ethanol.
Clear with xylene and mount with a resinous medium

Expected Results

Nuclei – blue black (Gallego) or red (Duprès)
Erythrocytes – orange
Cytoplasm – red
Muscle – light green
Bone – dark green
Collagen – blue

Notes

The celestine blue-hemalum sequence or Weigert’s iron hematoxylin may be suitable at step 2.
Solution B contains both a basic and an acid dye.
Other basic dyes may be substituted for toluidine blue:
- Methyl green 0.3 g
- Methylene blue 0.3 g
- Malachite green 0.2 g

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Duprès, (1935)
Journal number 11425, v. 46, pp. 77

Note: Gray identifies journals by a number from the world list of scientific periodicals, ed. 2, 1934. Unfortunately, the journal listed as number 11425 is not identified by name in the list he provides. In a second reference to the differentiator used in this technique he gives the journal number as 14425. This may be the intended reference:
Duprès, (1935)
Monitore zoologico italiano, v. 46, pp. 77
Florence, Italy.

March 30, 2023

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